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Biomedical subjects

G David

Publications and source records attributed to G David.

At least 73 records · Page 4Linked to original sources
PubMed2001

Developmental roles of the glypicans.

Glypicans are proteins with very characteristic structures that are substituted with heparan sulfate and that are linked to the cell surface via glycosylphosphatidylinositol. The modular structure of the glypicans has been highly conserved throughout evolution. Six glypicans have been identified so far in vertebrates. Mutations in Drosophila, humans and mice reveal a role for these cell surface molecules in the control of cell growth and differentiation. Their mechanism of action is not yet clear. Most likely, glypicans activate or determine the activity ranges of morphogens and growth factors such as FGFs, BMPs, Wnts, Hhs and IGFs.

Animals↗
PubMed2001

Heparan sulfate proteoglycan expression in cerebrovascular amyloid beta deposits in Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis (Dutch) brains.

Cerebrovascular deposition of amyloid beta protein (A beta) is a characteristic lesion of Alzheimer's disease (AD) and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D). Besides A beta, several other proteins and proteoglycans accumulate in cerebral amyloid angiopathy (CAA). We have now analyzed the expression of the heparan sulfate proteoglycan (HSPG) subtypes agrin, perlecan, glypican-1, syndecans 1-3 and HS glycosaminoglycan (GAG) side chains in CAA in brains of patients with AD and HCHWA-D. Hereto, specific well-characterized antibodies directed against the core protein of these HSPGs and against the GAG side chains were used for immunostaining. Glypican-1 was abundantly expressed in CAA both in AD and HCHWA-D brains, whereas perlecan and syndecans-1 and -3 were absent in both. Colocalization of agrin with vascular A beta was clearly observed in CAA in HCHWA-D brains, but only in a minority of the AD cases. Conversely, syndecan-2 was frequently associated with vascular A beta in AD, but did not colocalize with vascular A beta deposits in HCHWA-D. The three different syndecans, agrin, glypican-1 and HS GAG, but not perlecan, were associated with the majority of senile plaques (SPs) in all brains. Our results suggest a role for agrin in the formation of SPs and of CAA in HCHWA-D, but not in the pathogenesis of CAA in AD. Both syndecan-2 and glypican, but not perlecan, may be involved in the formation of CAA. We conclude that specific HSPG species may be involved in the pathogenesis of CAA in both AD and HCHWA-D, and that the pathogenesis of CAA and SPs may differ with regard to the involvement of HSPG species.

Agrin↗
PubMed2001

A 4-Mb BAC/PAC contig and complete genomic structure of the GPC5/GPC6 gene cluster on chromosome 13q32.

The glypicans compose a family of glycosylphosphatidylinositol-anchored heparan sulfate proteoglycans that may play a role in the control of cell division and growth regulation. So far, six members (GPC1-6) of this family are known in vertebrates. We report the construction of a high-resolution 4 Mb sequence-ready BAC/PAC contig of the GPC5/GPC6 gene cluster on chromosome region 13q32. The contig indicates that, like the GPC3/GPC4 genes on Xq26, GPC5 and GPC6 are arranged in tandem array. Both GPC5 and GPC6 are very large genes, with sizes well over 1 Mb. With a size of approximately 2 Mb, GPC5 would be the second largest human gene identified to date. Comparison of the long range gene organisation on 13q and Xq, suggests that these chromosomes share several regions of homology. Mutations and deletions affecting GPC3 are associated with the Simpson-Golabi-Behmel overgrowth syndrome. Mutational analysis of GPC5 and GPC6 in 19 patients with somatic overgrowth failed to reveal pathologic mutations in either of these genes, but identified several coding region polymorphisms.

Base Sequence↗
PubMed2001

Characterization of syntenin, a syndecan-binding PDZ protein, as a component of cell adhesion sites and microfilaments.

Syntenin is a PDZ protein that binds the cytoplasmic C-terminal FYA motif of the syndecans. Syntenin is widely expressed. In cell fractionation experiments, syntenin partitions between the cytosol and microsomes. Immunofluorescence microscopy localizes endogenous and epitope-tagged syntenin to cell adhesion sites, microfilaments, and the nucleus. Syntenin is composed of at least three domains. Both PDZ domains of syntenin are necessary to target reporter tags to the plasma membrane. The addition of a segment of 10 amino acids from the N-terminal domain of syntenin to these PDZ domains increases the localization of the tags to stress fibers and induces the formation of long, branching plasma membrane extensions. The addition of the complete N-terminal region, in contrast, reduces the localization of the tags to plasma membrane/adhesion sites and stress fibers, and reduces the morphotypical effects. Recombinant domains of syntenin with the highest plasma membrane localization display the lowest nuclear localization. Syndecan-1, E-cadherin, beta-catenin, and alpha-catenin colocalize with syntenin at cell-cell contacts in epithelial cells, and coimmunoprecipitate with syntenin from extracts of these cells. These results suggest a role for syntenin in the composition of adherens junctions and the regulation of plasma membrane dynamics, and imply a potential role for syntenin in nuclear processes.

Actin Cytoskeleton↗
PubMed2001

Efficient male and female germline transmission of a human chromosomal vector in mice.

A small accessory chromosome that was mitotically stable in human fibroblasts was transferred into the hprt(-) hamster cell line CH and developed as a human chromosomal vector (HCV) by the introduction of a selectable marker and the 3' end of an HPRT minigene preceded by a loxP sequence. This HCV is stably maintained in the hamster cell line. It consists mainly of alphoid sequences of human chromosome 20 and a fragment of human chromosome region 1p22, containing the tissue factor gene F3. The vector has an active centromere, and telomere sequences are lacking. By transfecting a plasmid containing the 5' end of HPRT and a Cre-encoding plasmid into the HCV(+) hamster cell line, the HPRT minigene was reconstituted by Cre-mediated recombination and expressed by the cells. The HCV was then transferred to male mouse R1-ES cells and it did segregate properly. Chimeras were generated containing the HCV as an independent chromosome in a proportion of the cells. Part of the male and female offspring of the chimeras did contain the HCV. The HCV(+) F1 animals harbored the extra chromosome in >80% of the cells. The HCV was present as an independent chromosome with an active centromere and the human F3 gene was expressed from the HCV in a human-tissue-specific manner. Both male and female F1 mice did transmit the HCV to F2 offspring as an independent chromosome with properties similar to the original vector. This modified small accessory chromosome, thus, shows the properties of a useful chromosomal vector: It segregates stably as an independent chromosome, sequences can be inserted in a controlled way and are expressed from the vector, and the HCV is transmitted through the male and female germline in mice.

Animals↗
PubMed2001

Syndecans serve as attachment receptors for human immunodeficiency virus type 1 on macrophages.

Macrophages are thought to represent one of the first cell types in the body to be infected during the early stage of human immunodeficiency virus type 1 (HIV-1) transmission and represent a potential viral reservoir in vivo. Thus, an understanding of HIV-1 attachment to these cells is fundamental to the development of novel anti-HIV-1 therapies. Although one of the major targets of HIV-1 in vivo--CD4(+) T lymphocytes--express high CD4 levels, other major targets such as macrophages do not. We asked in this study whether this low CD4 level on macrophages is sufficient to support HIV-1 attachment to these cells or whether cell surface proteins other than CD4 are required for this process. We show that CD4 alone is not sufficient to support the initial adsorption of HIV-1 to macrophages. Importantly, we find that heparan sulfate proteoglycans (HSPGs) serve as the main class of attachment receptors for HIV-1 on macrophages. Most importantly, we demonstrate that a single family of HSPGs, the syndecans, efficiently mediates HIV-1 attachment and represents an abundant class of attachment receptors on macrophages.

HIV Infections↗
PubMed2001

Mapping of the rat glypican genes.

The glypicans compose a family of glycosylphosphatidylinositol (GPI)-anchored heparan sulfate proteoglycans that play a role in the control of cell division and growth regulation. So far, six members (GPC1-6) of this family are known in vertebrates. The rat glypican gene 3 (Gpc3) was previously assigned to chromosome Xq36 (Shen et al., 1997). Using standard and radiation cell hybrids, we localized the five other rat glypican genes.

Animals↗
PubMed2001

[Value of RHD fetal genotyping in the prevention of anti-D immunization].

Anti-D prophylaxis is currently applied in France after birth of an RhD positive infant, after interruption of pregnancy and after some antenatal immunizing events (amniocentesis...). However this program does not cover all the prenatal exposures to fetal RhD antigen, and maternal Rh immunization continues to occur. DNA RhD genotyping of the fetus is now reliably performed on amniotic fluid, and pre diagnostic studies on fetal DNA extracted from maternal plasma are promising. The widespread use of fetal RhD genotyping on maternal blood would allow the antenatal administration of Rh immunoglobuline in all Rh negative patients bearing an Rh positive fetus, insofar as it would preclude exposing the other Rh negative patients to the above plasma derived and rather expensive blood product.

Female↗
PubMed2001

[The principle of precaution: its impact in medicine].

The principle of precaution first appeared about ten years ago as a new type of action and responsability in the environnemental domain. According to this priciple, immediate action must be taken to avoid serious environnemental consequences, without waiting for scientifically established proof of danger. Certain legalists are now recommending that this approach should be extended to the field of medicine. By recommending decisive action based on purely hypothetical risk, the principle of precaution goes well beyond classic preventive measures, which are introduced after identification and proof risk. This new approach is presented as an added safety precaution. Radical application of this concept could however lead to an irrational approach to health safety and constitute an obstacle to technical progress. Although the legal world has already adopted this new concept, it is important that the medical world contribute to evaluating this new standard.

Decision Making↗
PubMed2001

[Application of the principle of precaution to medicine].

The principle of precaution prescribes taking immediate action to avoid serious damage, rather than waiting for proof of the danger. It was extended to medical field, without taking account of the specificity of medicine, as well in individual medicine as in public health. Radical application of this concept could lead to serious repercussions. After analysis, it will considered as an expression of the historical confrontation between the science and the public opinion.

Defensive Medicine↗
PubMed2000

Characterization of the gene encoding the 100-kDa form of human 2',5' oligoadenylate synthetase.

The 2'-5' oligoadenylate synthetases (OAS) represent a family of interferon (IFN)-induced proteins implicated in the antiviral action of IFN. When activated by double-stranded (ds) RNA, these proteins polymerize ATP into 2'-5' linked oligomers with the general formula pppA(2'p5'A)n, n greater than or = 1. Three forms of human OAS have been described corresponding to proteins of 40/46, 69/71, and 100 kDa. These isoforms are encoded by three distinct genes clustered on chromosome 12 and exhibit differential constitutive and IFN-inducible expression. Here we describe the structural and functional analysis of the gene encoding the large form of human OAS. This gene has 16 exons with exon/intron boundaries that are conserved among the different isoforms of the human OAS family, reflecting the evolutionary link among them. The promoter region of the p100 gene is composed of multiple features conferring direct inducibility not only by IFNs but also by TNF and all-trans retinoic acid. In contrast, the induction of the p100 promoter by dsRNA is indirect and requires IFN type I production.

2',5'-Oligoadenylate Synthetase↗
PubMed2000

High molecular weight kininogen utilizes heparan sulfate proteoglycans for accumulation on endothelial cells.

Kininogens, the high molecular weight precursor of vasoactive kinins, bind to a wide variety of cells in a specific, reversible, and saturable manner. The cell docking sites have been mapped to domains D3 and D5(H) of kininogens; however, the corresponding cellular acceptor sites are not fully established. To characterize the major cell binding sites for kininogens exposed by the endothelial cell line EA.hy926, we digested intact cells with trypsin and other proteases and found a time- and concentration-dependent loss of (125)I-labeled high molecular weight kininogen (H-kininogen) binding capacity (up to 82%), indicating that proteins are crucially involved in kininogen cell attachment. Cell surface digestion with heparinases similarly reduced kininogen binding capacity (up to 78%), and the combined action of heparinases and trypsin almost eliminated kininogen binding (up to 85%), suggesting that proteoglycans of the heparan sulfate type are intimately involved. Consistently, inhibitors such as p-nitrophenyl-beta-d-xylopyranoside and chlorate interfering with heparan sulfate proteoglycan biosynthesis reduced the total number of kininogen binding sites in a time- and concentration-dependent manner (up to 67%). In vitro binding studies demonstrated that biotinylated H-kininogen binds to heparan sulfate glycosaminoglycans via domains D3 and D5(H) and that the presence of Zn(2+) promotes this association. Cloning and over-expression of the major endothelial heparan sulfate-type proteoglycans syndecan-1, syndecan-2, syndecan-4, and glypican in HEK293t cells significantly increased total heparan sulfate at the cell surface and thus the number of kininogen binding sites (up to 3. 3-fold). This gain in kininogen binding capacity was completely abolished by treating transfected cells with heparinases. We conclude that heparan sulfate proteoglycans on the surface of endothelial cells provide a platform for the local accumulation of kininogens on the vascular lining. This accumulation may allow the circumscribed release of short-lived kinins from their precursor molecules in close proximity to their sites of action.

Amino Acid Sequence↗
PubMed2000

Stimulation-evoked increases in cytosolic [Ca(2+)] in mouse motor nerve terminals are limited by mitochondrial uptake and are temperature-dependent.

Increases in cytosolic [Ca(2+)] evoked by trains of action potentials (20-100 Hz) were recorded from mouse and lizard motor nerve terminals filled with a low-affinity fluorescent indicator, Oregon Green BAPTA 5N. In mouse terminals at near-physiological temperatures (30-38 degrees C), trains of action potentials at 25-100 Hz elicited increases in cytosolic [Ca(2+)] that stabilized at plateau levels that increased with stimulation frequency. Depolarization of mitochondria with carbonylcyanide m-chlorophenylhydrazone (CCCP) or antimycin A1 caused cytosolic [Ca(2+)] to rise to much higher levels during stimulation. Thus, mitochondrial Ca(2+) uptake contributes importantly to limiting the rise of cytosolic [Ca(2+)] during repetitive stimulation. In mouse terminals, the stimulation-induced increase in cytosolic [Ca(2+)] was highly temperature-dependent over the range 18-38 degrees C, with greater increases at lower temperatures. At the lower temperatures, application of CCCP continued to depolarize mitochondria but produced a much smaller increase in the cytosolic [Ca(2+)] transient evoked by repetitive stimulation. This result suggests that the larger amplitude of the stimulation-induced cytosolic [Ca(2+)] transient at lower temperatures was attributable in part to reduced mitochondrial Ca(2+) uptake. In contrast, the stimulation-induced increases in cytosolic [Ca(2+)] measured in lizard motor terminals showed little or no temperature-dependence over the range 18-33 degrees C.

Action Potentials↗
PubMed2000

Myc-enhanced expression of Cul1 promotes ubiquitin-dependent proteolysis and cell cycle progression.

The c-Myc oncoprotein plays an important role in the growth and proliferation of normal and neoplastic cells. To execute these actions, c-Myc is thought to regulate functionally diverse sets of genes that directly govern cellular mass and progression through critical cell cycle transitions. Here, we provide several lines of evidence that c-Myc promotes ubiquitin-dependent proteolysis by directly activating expression of the Cul1 gene, encoding a critical component of the ubiquitin ligase SCF(SKP2). The cell cycle inhibitor p27(kip1) is a known target of the SCF(SKP2) complex, and Myc-induced Cul1 expression matched well with the kinetics of declining p27(kip1) protein. Enforced Cul1 expression or antisense neutralization of p27(kip1) was capable of overcoming the slow-growth phenotype of c-Myc null primary mouse embryonic fibroblasts (MEFs). In reconstitution assays, the addition of in vitro translated Cul1 protein alone was able to restore p27(kip1) ubiquitination and degradation in lysates derived from c-myc(-/-) MEFs or density-arrested human fibroblasts. These functional and biochemical data provide a direct link between c-Myc transcriptional regulation and ubiquitin-mediated proteolysis and together support the view that c-Myc promotes G(1) exit in part via Cul1-dependent ubiquitination and degradation of the CDK inhibitor, p27(kip1).

3T3 Cells↗
PubMed2000

Syntenin-syndecan binding requires syndecan-synteny and the co-operation of both PDZ domains of syntenin.

Syntenin is an adaptor-like molecule that binds to the cytoplasmic domains of all four vertebrate syndecans. Syntenin-syndecan binding involves the C-terminal part of syntenin that contains a tandem of PDZ domains. Here we provide evidence that each PDZ domain of syntenin can interact with a syndecan. Isolated or combined mutations of the carboxylate binding lysines in the inter-betaAbetaB loops and of the alphaB1 residues in either one or both the PDZ domains of syntenin all reduce syntenin-syndecan binding in yeast two-hybrid, blot-overlay, and surface plasmon resonance assays. PDZ2 mutations have more pronounced effects on binding than PDZ1 mutations, but complete abrogation of syntenin-syndecan binding requires the combination of both the lysine and the alphaB1 mutations in both the PDZ domains of syntenin. Isothermal calorimetric titration of syntenin with syndecan peptide reveals the presence of two binding sites in syntenin. Yet, unlike a tandem of two PDZ2 domains and a reconstituted PDZ1+PDZ2 tandem, a tandem of two PDZ1 domains and isolated PDZ1 or PDZ2 domains do not interact with syndecan bait. We conclude to a co-operative binding mode whereby neither of these two PDZ domains is sufficient by itself but where PDZ2 functions as a "major" or "high affinity" syndecan binding domain, and PDZ1 functions as an "accessory" or "low affinity" syndecan binding domain. The paired, but not the isolated PDZ domains of syntenin bind also strongly to the immobilized cytoplasmic domains of neurexin and B-class ephrins. By inference, these data suggest a model whereby recruitment of syntenin to membrane surfaces requires two compatible types of bait that are in "synteny" (occurring together in location) and engages both PDZ domains of syntenin. The synteny of compatible bait may result from the assemblies and co-assemblies of syndecans and other similarly suited partners in larger supramolecular complexes. In general, an intramolecular combination of PDZ domains that are weak, taken individually, would appear to be designed to detect rather than drive the formation of specific molecular assemblies.

Amino Acid Sequence↗